Ligand specific to β2-glycoprotein I and use thereof

ABSTRACT

The cholesterol derivative, represented by the following formula (1): 
                         
wherein R represents a C3–C23 saturated or unsaturated aliphatic hydrocarbon residue having an optional substituent, and, to the cholesterol backbone, —OH, —CHO, —COOH, —OOH, or an epoxy group may be added, specifically binds to β 2 -glycoprotein. By use of the derivative, β 2 -glycoprotein or a similar substance can be assayed in a very practical manner. Through the assay, a disease can be detected in a very practical manner.

This application is a U.S. national stage of International ApplicationNo. PCT/JP02/00723 filed Jan. 30, 2002.

TECHNICAL FIELD

The present invention relates to a ligand specific to β₂-glycoprotein I(β₂-GPI) and derivatives of the ligand; to an assay method for β₂-GPImaking use of any of the ligand and derivatives; to an assay method foran antibody recognizing a ligand-β₂-GPI complex; and to a method fordetecting a disease.

BACKGROUND ART

β₂-GPI is a major antigen which is present in patients withantiphospholipid syndrome (APS) and is recognized by “anantiphospholipid antibodies.” β₂-GPI is known to specifically bind tooxidized low-density lipoprotein (oxidized LDL; oxLDL), but not tonon-oxidized (native) low-density lipoprotein (LDL). PCT InternationalPatent Publication (pamphlet) WO 95/9363 discloses an oxLDL assay methodbased on a specific binding property with respect to β₂-GPI, a kit fordiagnosing an arteriosclerotic disease employing the assay method, etc.

However, the portion of the oxLDL structure which β₂-GPI recognizes forbinding has not been identified.

Therefore, identification of the portion of the oxLDL structure to whichβ₂-GPI specifically binds would realize an assay of β₂-GPI or a similarassay by use of an easier and simpler system employing a substanceincluding the portion. In addition, handling and storage of the reagentsused in the assay would be further facilitated, leading to provision ofconstant-quality assay reagents, assay kits, etc. at low cost.

In view of the foregoing, the present inventors have carried outextensive studies in order to provide a substance having a structurewhich specifically binds to β₂-GPI; a very practical assay method forβ₂-GPI or the like making use of the substance; and a very practicalmethod for detecting a disease on the basis of the assay method. Theinventors have isolated a substance having a structure whichspecifically binds to β₂-GPI, and based on the isolated substance, haveestablished an assay method for β₂-GPI or the like as well as a methodfor detecting a disease employing the assay method. The presentinvention has been accomplished on the basis of these findings.

DISCLOSURE OF THE INVENTION

Accordingly, the present invention is directed to a cholesterolderivative represented by the following formula (1) (hereinafterreferred to as the derivative of the present invention):

wherein R represents a C3–C23 saturated or unsaturated aliphatichydrocarbon residue having an optional substituent, and, to thecholesterol backbone, —OH, —CHO, —COOH, —OOH, or an epoxy group may beadded.

The aforementioned R preferably has one or more substituents selectedfrom the group consisting of —COOH, —OH, —CHO, an oxo group, and anepoxy group.

R is preferably a C3–C23 saturated or unsaturated fatty acid residuewhich may have one or more oxo groups. More preferably, R is a grouprepresented by HOOC—R′— (wherein R′ represents a C2–C22 saturated orunsaturated aliphatic hydrocarbon residue which may have one or more oxogroups).

R′ is preferably a linear-chain aliphatic hydrocarbon residue having oneoxo group.

In relation to specific examples of the derivative of the presentinvention, the invention is also directed to cholesterol derivativesrepresented by the following formulas (2) to (7).

The present invention is also directed to a solid phase on which thederivative of the present invention has been immobilized (hereinafterreferred to as the “solid phase of the present invention”).

The present invention is also directed to an assay method for β₂-GPI,characterized in that the method includes at least the following steps(hereinafter referred to as “assay method 1 of the present invention”):

a step of forming a complex of β₂-GPI and the cholesterol derivativeimmobilized on the solid phase of the present invention by bringing aspecimen into contact with the solid phase (Step 1); and

a step of detecting β₂-GPI contained in the complex which has beenformed in Step 1 (Step 2).

The present invention is also directed to an assay method for anantibody recognizing a “complex of β₂-GPI and the derivative of thepresent invention,” characterized in that the method includes at leastthe following steps (hereinafter referred to as “assay method 2 of thepresent invention”):

a step of forming a complex of the “complex of β-GPI and the cholesterolderivative immobilized on the solid phase of the present invention”(hereinafter, the complex of β-GPI and the cholesterol derivativeimmobilized on the solid phase of the present invention is referred toas the “β₂-GPI-cholesterol derivative complex”) and an antibodyrecognizing the β₂-GPI-cholesterol derivative complex by bringing β₂-GPIand a specimen into contact with the solid phase (Step 1); and

a step of detecting the antibody contained in the complex which has beenformed in Step 1 (Step 2).

The present invention is also directed to a method for detecting adisease (hereinafter referred to as the “detection method of the presentinvention”), characterized in that the method includes assaying anantibody recognizing the “complex of β₂-GPI and the derivative of thepresent invention” present in blood through the assay method 2 of thepresent invention and correlating the amount of the antibody present inblood to the disease. The disease is preferably an antiphospholipidsyndrome or thrombosis. The thrombosis is preferably arterialthrombosis.

The present invention is also directed to an assay kit for β₂-GPI,characterized in that the kit comprises at least the following (A) and(B) (hereinafter referred to as assay kit 1 of the present invention):

the solid phase of the present invention (A) and

a substance binding to β₂-GPI (B).

The present invention is also directed to an assay kit for an antibodyrecognizing “the complex of β₂-GPI and the derivative of the presentinvention,” characterized in that the kit comprises at least thefollowing (A) and (B) (hereinafter referred to as assay kit 2 of thepresent invention):

the solid phase of the present invention (A) and

a substance binding to an antibody recognizing “the complex of β₂-GPIand the derivative of the present invention” (B).

The assay kit 2 of the present invention is preferably a kit fordetecting a disease. The disease is preferably an antiphospholipidsyndrome or thrombosis. The thrombosis is preferably arterialthrombosis.

The present invention will next be described in detail. First,abbreviations used in the specification are listed below.

aPL: Antiphospholipid antibody

aCL: Anticardiolipin antibody

APS: Antiphospholipid syndrome

β₂-GPI: β₂-Glycoprotein I

CL: Cardiolipin

LDL: Low-density lipoprotein

oxLDL: Oxidized LDL

Cu²⁺-oxLDL: oxLDL formed through oxidation by CuSO₄

oxLig-1: Compound represented by formula (2),9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid, also called(7-ketocholesteryl-9-carboxynonanoate)

oxLig-2: Compound represented by formula (7),4,12-dioxo-12-(7-ketocholest-5-en-3β-yloxy)dodecanoic acid

13-COOH-7KC: Compound represented by formula (3),13-oxo-13-(7-ketocholest-5-en-3β-yloxy)tridecanoic acid

PS: Phosphatidylserine

SLE: Systemic lupus erythematosus

TLC: Thin-layer chromatography

HPLC: High-performance liquid chromatography

Chol: Cholesterol

DOPC: Dioleoylphosphatidylcholine

[³H]DPPC: L-3-phosphatidyl[N-methyl-³H]choline, 1,2-dipalmitoyl

DPPS: Dipalmitoylphosphatidylserine

PAPC: 1-Palmitoyl-2-arachidonoyl-phosphatidylcholine

In the present specification, numerals enclosed by “[ ]” refer inprinciple to mentioned document Nos. of the below-mentioned references.

<1> Derivative of the Present Invention

The derivative of the present invention is a cholesterol derivativerepresented by the following formula (1):

wherein R represents a C3–C23 saturated or unsaturated aliphatichydrocarbon residue having an optional substituent. Among thehydrocarbon residues, a C5–C20 hydrocarbon residue is preferred, aC8–C15 residue is more preferred, and a C9–C13 residue is particularlypreferred. To the cholesterol backbone, —OH, —CHO, —COOH, —OOH, or anepoxy group may be added. The aforementioned R preferably has one ormore substituents selected from the group consisting of —COOH, —OH,—CHO, an oxo group, and an epoxy group.

R is preferably a C3–C23 saturated or unsaturated fatty acid residuewhich may have one or more oxo groups. Among the fatty acid residues, aC5–C20 fatty acid residue is preferred, a C8–C15 residue is morepreferred, and a C9–C13 residue is particularly preferred.

R is preferably a group represented by HOOC—R′— (wherein R′ represents aC2–C22 saturated or unsaturated aliphatic hydrocarbon residue which mayhave one or more oxo groups). Among the groups represented by HOOC—R′—,a C4–C19 group is preferred, a C7–C14 group is more preferred, and aC8–C12 group is particularly preferred.

R′ is preferably a linear-chain aliphatic hydrocarbon residue, with alinear-chain aliphatic hydrocarbon residue having one oxo group beingmore preferred. The preferred number of carbon atoms of the aliphatichydrocarbon residue is the same as described above.

Specific examples of the derivative of the present invention include thecholesterol derivatives represented by the following formulas (2) to(7), respectively.

In the present specification, the substance represented by formula (3)is referred to as “13-COOH-7KC.”

In the present specification, the substance represented by formula (7)is referred to as “oxLig-2.”

The derivative of the present invention can be produced by isolating thesame from oxLDL through the method described in the below-mentionedexample. Alternatively, the derivative can be chemically synthesizedthrough the method described in the below-mentioned example.

The synthesized or isolated derivative of the present invention can beidentified through analyses such as NMR (¹H-NMR, ¹³C-NMR) analysis, massspectrometry, and analysis of binding performance to β₂-GPI.

<2> Solid Phase of the Present Invention

The solid phase of the present invention is a solid phase on which thederivative of the present invention has been immobilized.

No particular limitation is imposed on the solid phase employed forimmobilization of the derivative of the present invention thereon, solong as the solid phase can immobilize the derivative of the presentinvention thereon and is insoluble in water, a specimen, and a reactionmixture to be assayed. Examples of the form of the solid phase includeplates (e.g., wells of a microplate), tube, beads, membrane, and gel.Examples of the solid phase material include polystyrene, polypropylene,nylon, and polyacrylamide.

Of these, a plate made of polystyrene is preferred.

In order to immobilize the derivative of the present invention onto thesolid phase, a general immobilization method for lipid; e.g., thephysical adsorption method or the covalent bond method, can be employed.

Of these, the physical adsorption method is preferred, since the methodcan be carried out in a simple manner and is often employed in thefield.

In one specific mode of the physical adsorption method, the derivativeof the present invention is dissolved in a solvent, such as ethanol,methanol, or a mixture of methanol and chloroform; the solution isbrought into contact with the solid phase (e.g., a microplate); and thesolvent is evaporated, thereby adsorbing the derivative of the presentinvention on the solid phase.

The surface of the solid phase on which the derivative of the presentinvention has been immobilized may include a surface portion on whichthe derivative has not yet been immobilized. If β₂-GPI or anothermolecular species contained in a specimen is attached on such a surfaceportion, accurate assay results may fail to be obtained. Thus, theportion on which the derivative of the present invention has not beenimmobilized is preferably covered with a blocking substance added priorto contact between a specimen and the solid phase. Examples of theblocking substance include serum albumin, casein, skim milk, andgelatin, and commercial blocking substance products can also be used.

In one specific blocking method, a blocking substance (e.g., serumalbumin, casein, skim milk, or gelatin) is added to the solid phase, andthe solid phase is stored at about 37° C. for 30 minutes to 2 hours orat ambient temperature (15 to 25° C.) for 1 to 2 hours.

<3> Assay Method of the Present Invention

The assay method 1 of the present invention is an assay method forβ₂-GPI, characterized in that the method includes at least the followingsteps:

a step of forming a complex of β₂-GPI and the cholesterol derivativeimmobilized on the solid phase of the present invention by bringing aspecimen into contact with the solid phase (Step 1); and

a step of detecting β₂-GPI contained in the complex which has beenformed in Step 1 (Step 2).

The steps will be described individually.

Step 1:

The description set forth regarding the solid phase of the presentinvention is also applicable herein.

No particular limitation is imposed on the specimen which is to bebrought into contact with the solid phase of the present invention, solong as the specimen contains or may contain β₂-GPI, which is an assaytarget. Purification of the specimen in terms of β₂-GPI is optional, andmay not be performed. Specific examples of the specimen include blood,serum, and plasma. No particular limitation is imposed on the method ofbringing the specimen into contact with the solid phase of the presentinvention, so long as the molecules of the derivative of the presentinvention immobilized on the solid phase are brought into contact withthe β₂-GPI molecules contained in the specimen. Specifically, thespecimen may be added to the solid phase of the present invention so asto attain contact therebetween, or the solid phase of the presentinvention may be added to the specimen so as to attain contacttherebetween.

After contact between the specimen and the solid phase has beenattained, the system is preferably allowed to react for about one hourat, for example, 0 to 45° C., preferably 4 to 37° C., so as tosufficiently bind β₂-GPI contained in the specimen to the derivative ofthe present invention immobilized on the solid phase. After completionof reaction, preferably, the solid phase and the liquid phase aresufficiently separated from each other. Alternatively, washing of thesolid phase is preferably carried out. For example, the surface of thesolid phase is washed with a wash liquid, thereby removing non-specificadsorbed matter and non-reacted components present in the specimen.

Preferred examples of the wash liquid include buffers (e.g., phosphatebuffer, PBS, and Tris-HCl buffer) to which a non-ionic surfactant suchas a Tween series surfactant has been added.

Through contact between the specimen and the solid phase of the presentinvention, β₂-GPI contained in the specimen and the derivative of thepresent invention immobilized on the solid phase form a complex, wherebyβ₂-GPI contained in the specimen is fixed to the solid phase by themediation of the derivative of the present invention.

Step 2:

No particular limitation is imposed on the detection method for β₂-GPIincluded in the complex formed in Step 1. However, a substance whichbinds to β₂-GPI is preferably used.

Examples of the substance which binds to β₂-GPI include an antibodyrecognizing β₂-GPI. The antibody used herein may be a monoclonalantibody or a polyclonal antibody, and is appropriately selected inaccordance with the purpose of the β₂-GPI assay, required precision andsensitivity, etc. In general, when a monoclonal antibody, particularly amonoclonal antibody specifically recognizing β₂-GPI, is used, noisecaused by substances other than β₂-GPI can be reduced, and higherprecision and sensitivity can be attained as compared with the casewhere a polyclonal antibody is used.

The antibody against β₂-GPI can be produced through a routinepreparation method for a polyclonal antibody or a monoclonal antibodymaking use of β₂-GPI as an antigen. Alternatively, a known antibodyrecognizing β₂-GPI can also be used. Examples of such known monoclonalantibodies include EY2C9 (IgM) [36], WB-CAL-1 (IgG2a, κ) [37], andCof-22 (IgG1, κ) [38]. Characteristics of these antibodies will bedescribed in detail in the below-mentioned present example.

Such a “substance which binds to β₂-GPI” is preferably labeled with alabeling substance, from the viewpoint of easy detection.

Even when the “substance which binds to β₂-GPI” itself is not labeledwith a labeling substance, a labeled substance which binds to the“substance which binds to β₂-GPI” may also be used.

No particular limitation is imposed on the labeling substances employedfor the labeling, so long as the substances can label typical proteins.Examples include enzymes (e.g., peroxidase, alkalaine phosphatase,β-galactosidase, luciferase, and acetylcholine esterase), fluorescentdyes (e.g., fluorescein isothiocyanate (FITC)), chemical fluorescentsubstances (e.g., luminol), biotin, and avidin (including streptavidin).The labeling method is appropriately determined from known labelingmethods suited for the labeling substance; e.g., the glutaraldehydemethod, the periodate cross-linking method, the maleimide cross-linkingmethod, the carbodiimide method, and the activated ester method (seeChemistry of Proteins (part 2), published by Tokyo Kagaku Dojin, 1987).For example, when biotin is used as a labeling substance, a methodemploying a biotin hydrazide derivative (see Avidin-Biotin Chemistry: AHandbook, p. 57–63, published by PIERCE CHEMICAL COMPANY, 1994) isappropriately employed. When fluorescein isothiocyanate is used, amethod disclosed in Japanese Patent Publication (kokoku) No. 63-17843 ora similar method is appropriately employed.

In the case where an antibody (not labeled) against β₂-GPI is employedas the substance which binds to β₂-GPI, another antigen (labeled) whichbinds to the corresponding antibody (immunoglobulin) can be employed asa secondary antibody. More specifically, when EY2C9 is used, ananti-human-IgM antibody which has been labeled with a labeling substancecan be used, whereas when WB-CAL-1 or Cof-22 is used, an anti-mouse IgGantibody labeled with a labelling substance can be used. Examples of thelabeling substance include horseradish peroxidase (HRP). Commercialproducts of such a secondary antibody can be used.

By detecting a labeling substance which has been bound to β₂-GPI by themediation of the “substance which binds to β₂-GPI,” β₂-GPI included inthe complex formed in Step 1 can be detected.

The detection method can be appropriately determined by a person skilledin the art, in accordance with the labeling substance used. For example,when a peroxidase is employed as a labeling substance, hydrogen peroxideand a coloring substrate such as tetramethylbenzidine serving as asubstrate for the enzyme are added to the enzyme reaction system, andthe degree of coloring of the product can be detected by measuring thechange in absorbance. When a fluorescent substance or chemiluminescentsubstance is used, detection can be performed by measuring fluorescenceor luminescence provided from the solution after completion of reaction.

In the present specification, the term “detection” refers not only toqualitative detection (i.e., detection to check the presence or absenceof the substance to be detected), but also to quantitative detection(i.e., detection to determine the amount (concentration) of thesubstance to be detected). The same convention is also applied to theterm “assay” in the present specification.

When quantitative detection (assay) is performed, a calibration curverepresenting the relationship between the β₂-GPI concentration andcertain detected values (e.g., absorbance) of the standard substance isprepared in advance by use of a β₂-GPI standard solution of a knownconcentration. Then, a specimen having an unknown concentration of thesubstance to be detected is subjected to measurement, and theconcentration can be determined from the detected values by use of thecalibration curve.

The assay method 2 of the present invention can be provided by furthermodifying the assay method 1 of the present invention. In the assaymethod 2, a specimen is brought into contact with the “complex of β₂-GPIand the derivative of the present invention immobilized on the solidphase” formed in Step 1 of the assay method 1 of the present invention,and the antibody recognizing the “complex of β₂-GPI and the derivativeof the present invention” contained in the specimen is assayed.

Specifically, the assay method 2 is an assay method for an antibodyrecognizing the “complex of β₂-GPI and the derivative of the presentinvention,” characterized in that the method includes at least thefollowing steps:

a step of forming a complex of the “β₂-GPI-cholesterol derivativecomplex” and an antibody recognizing the “β₂-GPI-cholesterol derivativecomplex” by bringing β₂-GPI and a specimen into contact with the solidphase (Step 1); and

a step of detecting the antibody contained in the complex which has beenformed in Step 1 (Step 2).

The steps will be described individually.

Step 1:

The aforementioned description regarding the solid phase of the presentinvention is also applicable herein.

The β₂-GPI to be brought into contact with the solid phase of thepresent invention may or may not be completely purified, and the degreeof purification may be appropriately determined by a person skilled inthe art in accordance with factors such as the desired sensitivity.However, purified β₂-GPI is preferably used. Purification of β₂-GPI canbe performed through, for example, the method described in thebelow-mentioned example.

The same description as provided in relation to the assay method 1 ofthe present invention is also applied to the specimen to be brought intocontact with the solid phase of the present invention. The same contactmethod as employed in the assay method 1 of the present invention isapplied to the contact method for bringing β₂-GPI and the specimen intocontact with the solid phase of the present invention. No particularlimitation is imposed on the contact method, so long as the methodensures the chance to attain contact between β₂-GPI molecules and themolecules of the derivative of the present invention immobilized on thesolid phase of the present invention and the chance to attain contactbetween the “complex of β₂-GPI and the derivative of the presentinvention” and the antibody molecules present in the specimen andrecognizing the complex.

Similar to the assay method 1 of the present invention, after contactbetween the complex and the antibody has been attained, the system ispreferably allowed to react for about one hour at, for example, 0 to 45°C., preferably 4 to 37° C., so as to sufficiently bind β₂-GPI to thederivative of the present invention immobilized on the solid phase andalso to sufficiently bind the antibody present in the specimen to the“complex of β₂-GPI and the derivative of the present invention.” Thesame description as provided in relation to washing or other operationafter reaction performed in the assay method 1 of the present inventionis also applied herein.

By bringing the specimen and β₂-GPI into contact with the solid phase ofthe present invention, β₂-GPI and the derivative of the presentinvention immobilized on the solid phase form a complex, and theantibody recognizing the “β₂-GPI-cholesterol derivative complex”contained in the specimen binds to the complex, whereby the antibodypresent in the specimen is fixed to the solid phase by the mediation ofthe complex of β₂-GPI and the derivative of the present invention.

Step 2:

No particular limitation is imposed on the detection method for theantibody included in the complex formed in Step 1. However, preferably,a substance which binds to the antibody present in a specimen isemployed. Examples of the substance which binds to the antibody includean antibody recognizing an antibody (immunoglobulin) present in thespecimen. For example, in the case of assay of an antibody present inhuman serum (autoantibody), an anti-human-IgG antibody can be employedas a secondary antibody.

The description provided with respect to the substance which binds tothe antibody (e.g., secondary antibody) employed in the assay method 1of the present invention applies herein mutatis mutandis. Similar to theassay method 1 of the present invention, the substance which binds tothe antibody is preferably labeled with a labeling substance. Thedescriptions provided with respect to the assay method 1 of the presentinvention regarding the labeling substance employed as a label, thelabeling method, and the detection method for the labeling substanceapply herein mutatis mutandis.

<4> Detection Method of the Present Invention

The detection method of the present invention is a method for detectinga disease, characterized in that the method includes assaying anantibody present in blood and recognizing the “complex of β₂-GPI and thederivative of the present invention” through the assay method 2 of thepresent invention and correlating the amount of the antibody present inblood to the disease.

In the detection method of the present invention, firstly, an antibodyrecognizing the “complex of β₂-GPI and the derivative of the presentinvention” and present in blood is assayed through the assay method 2 ofthe present invention. The aforementioned assay method 2 of the presentinvention is also applied herein. Although the specimen used herein is a“blood,” the specimen is not necessarily a whole blood, and other bloodsamples may be used so long as the samples reflect the amount of theantibody present in blood. Specifically, there may also be used a plasmaor a serum derived from the blood, a diluted product thereof, or aproduct thereof modified within a degree so as not to affect theantibody present in the sample.

In the detection method of the present invention, secondly, a disease isdetected by correlating, to the disease, the amount (concentration) ofthe antibody present in such a specimen. The “amount of the antibody”may be the aforementioned antibody level (an actually measured value)obtained by use of the calibration curve which has been prepared on thebasis of the relationship between the concentration of the standardantibody product and certain detected values of the labeled substance.Alternatively, the “amount of the antibody” may be a ratio (a relativevalue) of the antibody level to the amount of antibody in blood of ahealthy subject (a human not suffering the disease to be detected) whichratio is obtained without using the calibration curve.

The aforementioned antibody level increases in the presence of a certaindisease. Therefore, when the blood antibody level is lower than that ofa healthy subject, such a low level can be correlated to the state of“suffering the disease” or the state of “highly likely suffering thedisease.” When the blood antibody level is equal to that of a healthysubject, the level can be correlated to the state of “not suffering thedisease” or the state of “less likely suffering the disease.”

In addition to check whether a subject suffers a certain disease or not,the detection method of the present invention includes detection of thedegree of suffering the disease. For example, in a person whoperiodically undergoes an assay of the blood antibody level, when theantibody level tends to increase, the tendency can be correlated to thestate of “progress of the disease” or the state of “highly likelyprogress of the disease.” In contrast, when the assayed antibody leveltends to decrease, the tendency can be correlated to the state of“amelioration of the disease” or the state of “highly likelyamelioration of the disease.” When the assayed antibody level isconstant, the constant amount can be correlated to the state of “nochange in sickness (healthiness)” or the state of “highly likely nochange in sickness (healthiness).”

The “disease” is preferably an antiphospholipid syndrome or thrombosis.The thrombosis is preferably arterial thrombosis.

<5> Kit of the Present Invention

The kit 1 of the present invention is an assay kit for β₂-GPI,characterized in that the kit comprises at least the following (A) and(B):

the solid phase of the present invention (A) and

a substance binding β₂-GPI (B).

The kit 2 of the present invention is an assay kit for an antibodyrecognizing “the complex of β₂-GPI and the derivative of the presentinvention,” characterized in that the kit comprises at least thefollowing (A) and (B):

the solid phase of the present invention (A) and

a substance binding to an antibody recognizing “the complex of β₂-GPIand the derivative of the present invention” (B).

The aforementioned description regarding the solid phase of the presentinvention is also applicable herein. The descriptions provided withrespect to the assay methods 1 and 2 of the present invention apply,mutatis mutandis, to the substance binding to β₂-GPI and to thesubstance binding to an antibody recognizing “the complex of β₂-GPI andthe derivative of the present invention.” No particular limitation isimposed on the constitution of the assay kits 1 and 2 of the presentinvention, so long as each kit contains the aforementioned (A) and (B).The kits may further include, as an additional component, for example, aspecies of known concentration serving as a standard for preparing acalibration curve (β₂-GPI (kit 1) and an antibody recognizing “thecomplex of β₂-GPI and the derivative of the present invention” (kit 2));a reagent for detecting a labeling substance; a reagent which labels thesubstance binding to β₂-GPI or which labels the substance binding to theantibody recognizing “the complex of β₂-GPI and the derivative of thepresent invention”; or a labeled substance thereof. In addition to theseadditional components, the kits may further contain, for example, ablocking substance, the aforementioned washing liquid, aspecimen-diluting agent, or an enzymatic reaction-stopping agent.

These kit components may be placed separately in individual containersand can be stored until use thereof as a kit which can be used inaccordance with the assay method of the present invention.

The assay of β₂-GPI by use of the kit 1 of the present invention can beperformed in accordance with the assay method 1 of the presentinvention, and the assay of the antibody recognizing “the complex ofβ₂-GPI and the derivative of the present invention” by use of the kit 2of the present invention can be performed in accordance with the assaymethod 2 of the present invention.

The kit 2 of the present invention is preferably a kit for detecting adisease. The disease is preferably an antiphospholipid syndrome orthrombosis. The thrombosis is preferably arterial thrombosis. In thiscase, the detection of a disease can be performed in accordance with thedetection method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Molecular interactions among β₂-GPI, LDLs, lipoproteins (PLs),and anti-β₂-GPI autoantibody, detected by an optical biosensor.

-   A: LDL (open square) or oxLDL (closed square) binding to solid phase    β₂-GPI;-   B: LDL (open square) or oxLDL (closed square) binding to solid phase    WB-CAL-1 in the presence of β₂-GPI;-   C: DOPE (open circle) or CL (closed circle) binding to solid phase    β₂-GPI; and-   D: DOPE (open circle) or CL (closed circle) binding to solid phase    WB-CAL-1 in the presence of β₂-GPI.

FIG. 2. Binding of β₂-GPI and anti-β₂-GPI autoantibodies to plasma LDLsor their lipid extracts:

-   A: Plasma LDLs, β₂-GPI, and mouse anti-human β₂-GPI monoclonal    antibody (Cof-22) were sequentially incubated in a plate coated with    Fab fragment of anti-apoB100 monoclonal antibody (1D2). Binding was    detected using HRP-labeled anti-mouse IgG.-   B to D: Lipid extracts from LDLs were applied to a plate. β₂-GPI    binding was detected using Cof-22 and using HRP-labeled anti-mouse    IgG (B). Subsequent binding of WB-CAL-1 (C) and EY2C9 (D) were    detected using HRP-labeled anti-mouse IgG (C), and with HRP-labeled    anti-human IgM (D), respectively. Open columns: without β₂-GPI,    closed columns: with β₂-GPI (C, D). Data are indicated as the mean±    SD of triplicate samples.

FIG. 3. Thin-layer chromatography (TLC) and ligand blot of lipidextracts from LDLs.

Lipids extracted from LDLs were spotted on silica gel plates anddeveloped in solvent A.

-   A: Staining with I₂ vapor, molybdenum blue, orcin/sulfuric acid, and    sulfuric acid/acetic acid as indicated in the figure.-   B: Ligand blot was performed with β₂-GPI and anti-β₂-GPI antibodies.    The region marked with an asterisk was scraped off and subjected to    further purification. Glu-Cer represents glucosylceramide, and    Gal-Cer represents galactosylceramide.-   C: Ligand blot of the scraped lipids (Band-1 and Band-2) was    performed by sequential treatment with β₂-GPI and anti-β₂-GPI    antibodies (2-step)-   D: Ligand blot of the eluate was performed by co-incubation of    β₂-GPI and anti-β₂-GPI antibodies (1-step).

FIG. 4. Elution profiles of Band-1 by reversed phase high performanceliquid chromatography (HPLC).

Scraped Band-1 was eluted on Sephacil-Peptide column and detected at 210nm (A) and 234 nm (B). Data of ligand blot analysis on eluate usingEY2C9 are also shown in (C).

FIG. 5. LC/MS of purified oxLig-1 and its methylated compound.

-   A: HPLC profiles of oxLig-1 (upper) and methylated oxLig-1 (lower)    at 234 nm.-   B: A positive ionization mass spectrum of 7-ketocholesterol.-   C: A negative ionization mass spectrum of oxLig-1.-   D: A positive ionization mass spectrum of oxLig-1.-   E: A negative ionization mass spectrum of methylated oxLig-1.-   F: A positive ionization mass spectrum of methylated oxLig-1.

FIG. 6. Synthesis of oxLig-1(9-oxo-9-(7-ketocholest-5-en-30β-yloxy)nonanoic acid) and itsmethylation.

FIG. 7. Nuclear magnetic resonance (NMR) spectrum of synthesized oxLig-1and its methylated compound. 300 MHz ¹H-NMR spectra of synthesizedoxLig-1 (A) and of methylated oxLig-1 (C). 75.3 MHz ¹³C-NMR spectra ofsynthesized oxLig-1 (B) and of methylated synthesized oxLig-1 (D).

FIG. 8. TLC and ligand blot analysis on synthesized oxLig-1.

-   A: oxLig-1 and synthesized oxLig-1 was spotted on a TLC plate,    developed with solvent A, and detected with I₂-vapor.-   B: Ligand blot of synthesized oxLig-1 was performed with anti-β₂-GPI    antibodies in the presence (+) or absence (−) of β₂-GPI.

FIG. 9. LC/MS of synthesized oxLig-1 and its methylated compound.

-   A: LC chromatograms of synthesized oxLig-1 at 210 nm and at 234 nm.-   B: A positive ionization mass spectrum of synthesized oxLig-1.-   C: A negative ionization mass spectrum of synthesized oxLig-1.-   D: LC chromatograms of methylated synthesized oxLig-1 at 210 nm and    at 234 nm.-   E: A positive ionization mass spectrum of methylated oxLig-1.-   F: A negative ionization mass spectra of methylated synthesized    oxLig-1.

FIG. 10. Effect of phosphatidylserine (PS) or oxLig-1 content on thebinding of liposomes to macrophages. A monolayer of J774.A1 cells wasincubated for 2 hours at 4° C. with Celgrosser-P medium containing³H-labeled liposomes (50 nmol lipid/well). Open circle representsPS-liposomes; closed circle represents PS-Chol-liposomes; open squarerepresents oxLig-1-liposomes; and closed square representsoxLig-1-Chol-liposomes. Data are indicated as the mean± SD of triplicatesamples.

FIG. 11. β₂-GPI and anti-β₂-GPI monoclonal antibody-dependent binding ofligand-containing liposomes to macrophage. A monolayer of J774.A1 cellswas incubated for 2 hours at 4° C. with Celgrosser-P medium containing³H-labeled liposomes (50 nmol lipid/well) and WB-CAL-1, in the presenceor absence of β₂-GPI (200 μg/ml).

-   A: Binding of PS-liposomes (PS: 50 mol %,) to J774.A1. cells in the    presence (closed square) or absence (open square) of β₂-GPI (200    μg/ml)-   B: Binding of oxLig-1-liposomes (oxLig-1: 40 mol %) to J774.A1 cells    in the presence (black bar) or absence (white bar) of β₂-GPI (200    μg/ml).-   C: A monolayer of J774.A1 cells was incubated for 2 hours at 4° C.    with Celgrosser-P medium containing ³H-labeled synthesized    oxLig-1-liposomes (50 nmol lipid/well) and WB-CAL-1, in the presence    or absence of β₂-GPI (200 μg/ml). Binding of synthesized    oxLig-1-liposomes in the absence of β₂-GPI and WB-CAL-1 (open    circle), binding of synthesized oxLig-1-liposomes in the presence of    β₂-GPI (open square), binding of synthesized oxLig-1-liposomes in    the presence of WB-CAL-1 (closed square), and binding of synthesized    oxLig-1-liposomes in the presence of both β₂-GPI and WB-CAL-1    (closed circle). Data are indicated as the mean ± SD of triplicate    samples.

FIG. 12. Autoantibodies present in APS serum samples against solid phaseβ₂-GPI-oxLig-1 complex. Serum samples were obtained from healthysubjects (n=24) and APS patients with episodes of thrombosis (n=52).

-   A: Antibody values in individual serum samples-   B: Relationship between anti-β₂-GPI-oxLig-1 antibody values and    β₂-GPI-dependent aCL values-   C: Relationship between anti-β₂-GPI-oxLig-1 antibody values and    anti-β₂-GPI antibody values.

FIG. 13. Relationship between anti-β₂-GPI-oxLig-1 antibody values andantibody values of the β₂-GPI-dependent aCL (anti-β₂-GPI-CL antibody) inELISA, and relationship between anti-β₂-GPI-oxLig-1 antibody values andanti-β₂-GPI antibody values in ELISA. Plasma samples of 133 APS and/orSLE patients (87 APS patients and 47 SLE only patients) were assayedthrough the method described in relation to materials and methods.

FIG. 14. Relationship between antibody values and clinical episodes.Plots of anti-β₂-GPI-oxLig-1 antibody values of APS and/or SLE patients(without clinical episodes (open circles), with clinical episodes (graycircles)). The values of p represent Mann-Whitney U-test results. Thedashed lines represent a cut-off value (the level exceeding the averaged(healthy control) antibody value by 3×SD).

FIG. 15. TLC and Ligand blot profiles of oxLig-2, Me-oxLig-2,13-COOH-7KC, and Me-13-COOH-7KC.

FIG. 16. Elution profiles of Band-2 obtained by reversed phase highperformance liquid chromatography (HPLC).

Scraped Band-2 was eluted on Sephacil-Peptide column and detected at 210nm (A) and 234 nm (B). Data of ligand blot analysis on eluate usingEY2C9 are also shown in (B). Fraction 14 was purified again through HPLCunder the same conditions so as to confirm the purity (C and D).

FIG. 17. LC/MS of purified or synthesized β₂-GPI ligands. Positiveionization mass spectra of oxLig-1 (A), oxLig-2 (C), and 13-COOH-7KC (E)(left column) and negative ionization mass spectra of oxLig-1 (B),oxLig-2 (D), and 13-COOH-7KC (F) (right column).

FIG. 18. Structures of cholesteryl esters serving as β₂-GPI ligands.

Structures of cholesteryl linoleate (A), oxLig-1 (B), oxLig-2 (C), and13-COOH-7KC (E). Schemes of fragmentation possibly occurring in massspectroscopy are specified by means of arrows. Scission at each arrowposition will result in formation of the corresponding fragment ion “D.”

FIG. 19. Direct binding of ligand-containing liposomes to macrophage.

A monolayer of J774.A1 cells was incubated for 2 hours at 4° C. withCelgrosser-P medium containing [³H]-labeled liposomes (containing apredetermined ligand, 50 nmol lipid/well). DPPS-containing liposomes(open squares), oxLig-1-containing liposomes (closed squares),oxLig-2-containing liposomes (closed circles), and13-COOH-7KC-containing liposomes (closed triangles). Data are indicatedas mean± SD of triplicate samples.

FIG. 20. β₂-GPI and anti-β₂-GPI antibody-dependent binding ofligand-containing liposomes to macrophage.

A monolayer of J774.A1 cells was incubated for 2 hours at 4° C. withCelgrosser-P medium containing [³H]-labeled liposomes (containing apredetermined ligand (30 mmol %), 50 nmol lipid/well) and in thepresence (black bars) or absence (white bars) of β₂-GPI (200 μg/mL) andWB-CAL-1 (200 μg/mL).

Chart A: Cholesteryl linoleate-containing liposomes; Chart B:oxLig-1-containing liposomes, Chart C: oxLig-2-containing liposomes; andChart D: 13-COOH-7KC-containing liposomes. In Charts C and D, comparisonwas made with methylated ligand-containing liposomes. Data are indicatedas mean± SD of triplicate samples.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will next be described in more detail by way ofexample, which should not be construed as limiting the inventionthereto.

<1> Materials and Methods

Firstly, materials and methods employed in the present example will bedescribed.

(1) Chemicals

L-α-Dipalmitoylphosphatidylserine (DPPS), CL, Chol, and7-ketocholesterol (5-cholesten-3β-ol-7-one) were obtained from SigmaChemical Co. PAPC and DOPC from Avanti Polar Lipids Inc. [³H]DPPC (80Ci/mmol) from Amersham-Pharmacia Biotech. Other chemicals were obtainedfrom commercial sources and of reagent grade quality.

(2) Purification of Human β₂-GPI

β₂-GPI was purified from normal human plasma as described in [35] withslight modification. Pooled plasma from healthy subjects werechromatographed on a heparin-Sepharose column (or CL-polyacrylamide gelcolumn), on a DEAE-cellulose column, and on an anti-β₂-GPI affinitycolumn. To remove any contamination by IgGs, the β₂-GPI-containingfraction was further passed through a protein A-Sepharose column. Thefinal β₂-GPI fraction was delipidated by extensive washing withn-butanol.

(3) Anti-β₂-GPI Positive Serum Samples were Obtained from APS Patientswith Episodes of Arterial Thrombosis.

To eliminate endogenous β₂-GPI in some experiments, serum samples werepassed through a heparin-Sepharose column. The effluent was dialyzedagainst PBS and was used for ELISAs.

(4) Monoclonal Antibodies (mAbs)

EY2C9 (IgM): A human monoclonal anti-β₂-GPI autoantibody, establishedfrom peripheral blood lymphocytes from an APS patient [36].

WB-CAL-1 (IgG2a, κ): A mouse monoclonal anti-β₂-GPI autoantibody,derived from an (NZW×BXSB) F1 mouse [37].

Cof-22 (IgG1, κ): A monoclonal anti-human β2-GPI antibody, establishedfrom BALB/c mice immunized with human β₂-GPI [38].

Both EY2C9 and WB-CAL-1 bind either to a complex of β₂-GPI and CL and toβ₂-GPI adsorbed on an oxygenated polystyrene plates. In contrast, Cof-22is specific for human β₂-GPI and recognizes its native structure.

(5) Preparation of oxLDL and Lipid Extraction

Plasma LDL (1.019<d<1.063 g/mL) was isolated by ultracentrifugation fromfresh normal human plasma, as described in [39]. LDL (100 μg protein/mL)was oxidized by incubating with 5 μM CuSO₄ (PBS solution) for 8 hours at37° C. To stop the oxidation, 1 mM EDTA was added. The oxidized samplewas extensively dialyzed against PBS containing 1 mM EDTA.

Protein concentration was determined using BCA protein assay reagent(product of Pierce Chemical Co.). An aliquot of a sample was taken todetermine thiobarbituric acid reactive substance (TBARS) value servingas an index of an extent of oxidation [40], and for use in agarose gelelectrophoresis. A lipid fraction was extracted from LDLs according tothe method described in [41].

(6) Assay for Molecular Interaction

Real-time molecular analysis was performed using an optical biosensor,IAsys (product of Affinity Sensors).

Binding of β₂-GPI to LDLs or liposomes: Biotinyl-β₂-GPI was immobilizedon a biotinyl-cuvette via streptavidin. LDLs or liposomes at variousconcentrations were placed in the cuvette. Antibodies binding to LDLs orliposomes: The biotinyl-WB-CAL-1 was immobilized on the cuvette. LDLs orliposomes were added at various concentrations in the presence (10μg/mL) or absence of β₂-GPI.

(7) ELISA for Detecting Binding of β₂-GPI to Anti-β₂-GPI Antibody

Binding to LDLs: A microtiter plate (Immulon 2HB, Dynex TechnologiesInc.) was coated with 50 μL of F(ab′)2 of 1D2 (anti-apoB 100 antibody;10 μg/mL, product of Yamasa Corp.) by incubation overnight at 4° C.After blocking with PBS containing 1% skim milk, the plate was incubatedwith LDLs for one hour. The wells were incubated sequentially withβ₂-GPI (15 μg/mL), Cof-22, and horseradish peroxidase (HRP)-labeledanti-mouse IgG, each for one hour. The color was developed with H₂O₂ ando-phenylenediamine, and OD was measured at 490 nm.

Binding to extracted lipids: A microtiter plate (Immulon 1B, DynexTechnologies Inc.) was coated with lipids extracted from LDLs (50 μg/mL,50 μL/well) by ethanol evaporation. The wells were blocked with PBScontaining 1% BSA for one hour and the wells were incubated with 30μg/mL of β₂-GPI for one hour. Then, β₂-GPI binding was detected usingCof-22 and HRP-labeled anti-mouse IgG antibodies. Alternatively, ananti-β₂-GPI autoantibody (Cof-22, WB-CAL-1, or EY2C9) was diluted with a0.3% BSA-containing PBS, and the diluted antibody was added to thecoated plate (1.0 μg/mL, 100 μL/well) and incubated with β₂-GPI (15μg/mL) for one hour. The binding of the antibody was detected by anHRP-labeled anti-mouse IgG /or an anti-human IgM. The color wasdeveloped with H₂O₂ and o-phenylenediamine, and OD was measured at 490nm. Between these steps, extensive washing were done using PBScontaining 0.05% Tween 20.

(8) Thin Layer Chromatography (TLC) and Ligand Blot Analysis

Extracted lipids were spotted on a Polygram silica gel plate (product ofMachery-Nagel) and developed in chloroform/methanol/30% ammonia/water(120:80:10:5, v/v/v/v, hereinafter referred to as solvent A). The platewas stained with I₂ vapor, or with a spray of molybdenum blue, of 2Nsulfuric acid containing 2% orcin, or of glacial acetic acid/sulfuricacid (19:1, v/v) (Lieberman-Burchard reaction). Alternatively, thedeveloped plate was subjected to ligand blot with β₂-GPI and ananti-β₂-GPI antibody. TLC plates were blocked with PBS containing 1%bovine serum albumin (BSA) and were subsequently incubated with β₂-GPI,anti-β₂-GPI antibodies (Cof-22, WB-CAL-1, or EY2C9), and HRP-labeledanti-mouse IgG antibodies or anti-human IgM antibodies for one hour. Ineach step, plates were extensively washed with PBS. The color wasdeveloped with H₂O₂ and 4-methoxy-1-naphtol (product of Aldrich). Theligand-enriched bands scraped from the TLC plate were subjected toanother TLC in chloroform/methanol (8:1, v/v) (hereinafter referred toas solvent B). For large scale purification of the ligand, extractedlipids were loaded on a TLC silica gel 60 plate (PLC plate; product ofMerck) of 2 mm thickness.

(9) HPLC

A fraction rich in a ligand (originating from oxLDL) obtained bytwo-step TLC was analyzed by reversed phase HPLC on a Sephasil PeptideC-18 5-μm column (4.6 mm×250 mm; product of Amersham-Pharmacia Biotech).The column underwent elution with a mixture ofacetonitrile/isopropanol/water (60:30:2, v/v/v, hereinafter referred toas solvent C) at a flow rate of 0.5 mL/min and fractionated everyminute. Each eluate was spotted on a TLC plate and subjected to ligandblot with β₂-GPI and EY2C9.

(10) NMR

¹H-NMR and ¹³C-NMR spectra were recorded by means of ASX-300spectrometer (Bruker).

(11) Mass Spectroscopy

Synthesized oxLig-1 was analyzed on a Shim-pack VP-ODS column (4.6mm×150 mm) with an LC/MS-QP8000α (Shimadzu Corp.), using solvent C(LC/MS; liquid chromatography/mass spectroscopy). Positive and negativeionization mass spectra were recorded in the mass range of 50–850.

Mass spectrometry of oxLig-1, oxLig-2 purified from Band-2, andsynthesized 13-COOH-7KC, which are β₂-specific ligands, was carried outon a Shim-pack FC-ODS column (4.6 mm×30 mm) with an LC/MS-2010spectrometer (Shimadzu Corp.), using solvent F (30% acetone in methanol)and water with a linear gradation in concentration (50 to 100%).Positive and negative ionization mass spectra were taken in a mass rangeof 50 to 750.

The field desorption (FD) mass spectra of synthesized oxLig-1 andmethylated oxLig-1 were recorded on JMS-SX102A and JMS AX-500spectrometers (product of JEOL), respectively.

(12) Synthesis of β₂-GPI-Specific Ligand (oxLig-1)

The ligand, oxLig-1, was first isolated from Cu²⁺-oxLDL through twosteps of TLC, followed by HPLC, and finally identified as9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid(7-ketocholesteryl-9-carboxynonanoate) (FIG. 1). In the present example,the synthesized oxLig-1 was used instead of purified oxLig-1. To asolution of 7-ketocholesterol (5-cholesten-3β-ol-7-one, 50.1 mg, 0.125mmol) and azelaic acid (70.6 mg, 0.375 mmol) in acetone (4 mL) wereadded 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (95.8mg, 0.50 mmol) and 4-(dimethylamino)pyridine (30.5 mg, 0.25 mmol). Themixture was stirred at room temperature for two days, concentrated, andextracted with chloroform. The extract was successively washed with 2Mhydrochloric acid and dried over magnesium sulfate anhydrate, and thesolvent was evaporated. The residue was subjected to columnchromatography on silica gel using toluene/ethyl acetate (3:1, v/v), tothereby yield oxLig-1 (36.0 mg, yield: 50.4%).

(13) Analysis Results of Synthesized oxLig-1

(NMR); ¹H-NMR (300 MHz, CDCl₃): δ=5.71 (s, ¹H, H-6), 4.78–4.69 (m, ¹H,H-3); ¹³C-NMR (75.5 MHz, CDCl₃): δ=202.5, 179.7, 173.4, 164.5, 127.1,72.4, 55.2, 50.4, 50.2, 45.8, 43.5, 39.9, 38.7, 36.6, 36.1, 29.2, 28.9,28.4, 25.3, 25.0, 24.2, 23.2, 23.0, 19.3, 17.7, 12.4. (Massspectroscopy) m/z (FD-MS): 571 [(M+H)⁺, C₃₆H₅₉O₅]

(14) Methylation of oxLig-1

To a solution of purified or synthesized oxLig-1 (7.5 mg, 0.0131 mmol)in ether (2 mL), a diazomethane-ether solution was added at roomtemperature, and the mixture was stirred for 2 hours. Acetic acid wasadded to the solution while stirring. One day after addition of aceticacid, the solvent was evaporated to give methylated oxLig-1 preparation.

Hereinafter, a methylated species may be represented with a prefix“Me-.”

(15) Analysis Results of Methylated Synthesized oxLig-1

(NMR); ¹H-NMR (300 MHz, CDCl₃): d=5.71 (s, ¹H, H-6), 4.78–4.69 (m, m,¹H, H-3), 3.67 (s, ¹H, COOCH3); ¹³C-NMR (75.5 MHz, CDCl₃): d=202.4,174.7, 173.4, 164.4, 127.1, 72.4, 55.2, 50.4, 45.8, 43.5, 39.9, 38.7,36.6, 36.4, 36.1, 29.3, 28.4, 26.7, 25.3, 24.2, 23.2, 23.0, 21.6, 19.3,17.7, 12.4. (Mass spectroscopy) m/z (FD-MS): 585 [(M+H)⁺, C₃₇H₅₁O₅]

(16) Methylation of oxLig-1

Under cooling with ice, 1-methyl-3-nitro-1-nitrosomethylguanidine (0.20g) was added to a mixture of 2M sodium hydroxide (10 mL) and diethylether (10 mL). The resultant mixture was stirred for several minutes,whereby a pale-yellow liquid was separated as an upper layer. Thethus-separated liquid was employed for methylation. A diazomethanesolution (2 mL) was added dropwise to a solution (1 mL) of a lipidligand (oxLig-2 or 13-COOH-7KC) (1.0 mg) in diethyl ether at 0° C. Eachof the formed two liquids was stored overnight in a refrigerator. TLCanalysis revealed that each starting substance was completely converted.The solvent of each solution was removed through air blow, to therebyyield a methyl ester of each ligand as a white, amorphous matter.

(17) Synthesis of 13-COOH-7KC

WSC (95.8 mg, 50 mmol) and DMAP (30.5 mg, 0.25 mmol) were added to anacetone solution (4 mL) containing 7-ketocholesterol(5-cholesten-3β-ol-7-one) (50.1 mg, 0.13 mmol) and tridecanedioic acid(brassylic acid) (61.8 mg, 0.25 mmol). The mixture was stirred at roomtemperature for two days, and the reaction mixture was concentrated andextracted with chloroform. The extract was sequentially washed with 2Mhydrochloric acid, an aqueous saturated sodium hydrogencarbonatesolution, and brine. The thus-obtained liquid was dried over magnesiumsulfate anhydrate, and the solvent was evaporated. The residue wasapplied to silica gel chromatography using toluene/ethyl acetate (3:1,v/v), to thereby yield 44 mg of 13-COOH-7KC (yield: 56.0%). NMR spectraand a mass spectrum (FD-MS) of the product were recorded in the samemanner as described above.

(NMR); ¹H-NMR (300 MHz, CDCl₃): δ=5.69 (s, ¹H, H-6), 4.80–4.67 (m, ¹H,H-3); ¹³C-NMR (75.5 MHz, CDCl₃): δ=202.2, 179.6, 173.1, 164.7, 126.9,72.3, 55.3, 50.5, 50.1, 45.3, 43.5, 40.6, 39.2, 38.6, 36.5, 36.0, 29.1,28.8, 28.3, 25.2, 24.9, 24.1, 23.1, 22.9, 19.2, 17.6, 12.3 (Massspectroscopy) m/z (FD-MS): 627 [(M+H)⁺, C₄₀H₆₇O₅]

(18) Preparation of Liposomes

Liposomes were prepared through a method as described in [42], with thefollowing lipid compositions. Lipid proportions by mol of 0, 10, 25, and50% PS-liposomes are as follows.

DOPC/DPPS/[³H]-DPPC (80 Ci/mmol)

-   0%: 100/0/0.00225-   10%: 90/10/0.00225-   25%: 75/25/0.00225-   50%: 50/50/0.00225

Lipid proportions by mol of 0, 5, 12.5, and 25% PS-Chol-liposomes are asfollows.

DOPC/DPPS/Chol/ [³H]-DPPC (80 Ci/mmol)

-   0%: 50/0/50/0.00225-   5%: 45/5/50/0.00225-   12.5%: 37.5/12.5/50/0.00225-   25%: 25/25/50/0.00225

Lipid proportions by mol of 0, 10, 20, and 40% oxLig-1 (purifiedproduct)-liposomes are as follows.

DOPC/oxLig-1/[³H]-DPPC (80 Ci/mmol)

-   5%: 100/0/0.00225-   10%: 90/10/0.00225-   20%: 80/20/0.00225-   40%: 60/40/0.00225)

Lipid proportions by mol of 0, 5, 10, 20, 30, and 40% oxLig-1 (purifiedproduct)-Chol-liposomes are as follows.

DOPC/Chol/oxLig-1/[³H]-DPPC (80 Ci/mmol)

-   50%: 50/50/0/0/0.00225-   5%: 50/45/5/0.00225-   10%: 50/40/10/0.00225-   20%: 50/30/20/0.00225-   30%: 50/20/30/0.00225-   40%: 50/10/40/0.00225

oxLig-1 (synthesized product)-Liposomes were prepared using synthesizedoxLig-1 in a manner similar to preparation of oxLig-1 (purifiedproduct)-liposomes. A mixture of the desired lipids inchloroform/methanol (1:1, v/v) was placed in a pare-shaped flask and thesolvent was removed in a rotary evaporator under reduced pressure. Thedried lipids were dispersed with a mixer in 0.3M glucose solution. Thenthe liposome solution was sonicated for 5 minutes at 70° C. with aprobe-type sonicator.

Liposomes of oxLig-2, 13-COOH-7KC, etc. were also prepared in a similarmanner by use of the following lipid compositions. Liposomes containingeach ligand in an amount of 0, 10, 25, 30, and 50% were prepared by useof a DOPC/ligand/[³H]-DPPC (80 Ci/mmol). The amount of [³H]-DPPC usedwas 0.225%. As the ligands, cholesteryl linoleate, DPPS, oxLig-1,oxLig-2, methylated oxLig-2 (Me-oxLig-2), 13-COOH-7KC, and methylated13COOH-7KC (Me-13COOH-7KC) were used.

(19) Cell Culture and Liposome Binding

A monolayer culture of murine macrophage-like cells (J774A.1) obtainedfrom Riken Cell Bank (Tsukuba) was maintained in RPMI 1640 mediumsupplemented with 10% fetal carf serum (FCS). For binding experiments,the cells (8×10⁵ cells/mL, RPMI1640) were dispensed in an amount of 1mL/well into a 12-well culture plate (Sumitomo Bakelite Co., Ltd.) andwere incubated for 24 hours at 37° C., then the culture broth wasreplaced with Celgrosser-P medium (Sumitomo Pharmaceutical Co.). Afterone hour of preincubation at 37° C., 50 μL of liposomes (50 nmollipid/well) with or without β₂-GPI (200 μg/mL) and WB-CAL-1 (100 μg/mL)were added to each culture, and the cells were incubated at 4° C. and/or37° C. The wells were next washed with chilled PBS, and the cells werelysed by adding 1 mL of 0.1N NaOH. An aliquot was taken fordetermination of cellular proteins and of radioactivity associated withthe cells. Protein concentration was determined in a manner as describedabove.

(20) Anti-β₂-GPI ELISA (ELISA of IgG Autoantibody Against β₂-GPI)

Anti-β₂-GPI ELISA (ELISA of IgG autoantibody against β₂-GPI) wasperformed through a method as described in [43]. Briefly, β₂-GPI (10μg/mL, 50 μL/well) was adsorbed on an oxygenated polystyrene plate(carboxylated, Sumilon C-type, Sumitomo Bakelite Co., Ltd.) byincubating overnight at 4° C. The plates were blocked with 3% gelatin,and 100 μL/well of anti-β₂-GPI monoclonal antibody or of 100-folddiluted plasma samples was added to the plate, followed by incubationfor one hour. A antibody binding to β₂-GPI was probed using HRP-labeledanti-human IgG or IgM or anti-mouse IgG. The color was developed withH₂O₂ and o-phenylenediamine, and OD was measured at 490 nm. Betweenthese steps, extensive washing were done using PBS containing 0.05%Tween 20.

(21) ELISA for Antibodies Against a Protein-oxLig-1 Complex

Synthesized oxLig-1 (50 μg/mL, 50 μL/well) was adsorbed by evaporationon a polystyrene plate (Immulon 1B; Dynex Technologies Inc.), and theplate was then blocked with 1% BSA. Serum samples (diluted 1:100 withPBS containing 0.3% BSA) were incubated in the wells with β₂-GPI (15μg/mL) or other proteins for one hour at room temperature. Antibodybinding was detected with HRP-labeled anti-human IgG. Further steps wereperformed through a method as described in “anti-β₂-GPI ELISA.”

(22) β₂-GPI-Dependent aCL

CL (50 μg/mL, 50 μL/well) was adsorbed on a polystyrene plate (Immulon1B), and further steps were performed through a method as described in“ELISA for antibodies against a protein-oxLig-1 complex.”

(23) Statistical Analysis

Statistical analysis was performed by use of StatView software (productof Abacus Concepts). Comparative studies between the autoantibody andthe clinical episode were carried out by means of the Fisher's exacttest.

The relationship between the antibody value and the clinical episode wasevaluated by means of the Mann-Whitney U-test.

<2> Results

(1) Molecular Interaction

oxLDL but not native LDL showed highly specific binding to β₂-GPI (FIG.1A). A dose-dependent binding of oxLDL to solid phase WB-CAL-1 wasobserved only in the presence of β₂-GPI (10 μg/mL), and no specificbinding of native LDL was observed (FIG. 1B). In a control experiment,CL showed large extent of binding, while DOPE did not show any specificbinding (FIG. 1C). CL also showed dose-dependent binding to WB-CAL-1 inthe presence of β₂-GPI, while DOPE did not (FIG. 1D).

(2) Binding of β₂-GPI and Anti-β₂-GPI Antibody to Solid Phase LDLs orLipids Derived therefrom

β₂-GPI specifically bound with high avidity to immobilized oxLDL, butminimally to native LDL (FIG. 2A). Lipids were extracted from LDLs,immobilized on a plate, and subjected to binding assays for β₂-GPI bydetecting with Cof-22 monoclonal antibody and anti-β₂-GPI antibodies(i.e., WB-CAL-1 and EY2C9). The assays showed specific binding of β₂-GPI(FIG. 2B) and of β₂-GPI-mediated antibody to the lipids derived fromoxLDL (FIGS. 2C and 2D), but did not to those derived from native LDL(FIGS. 2B–2D).

(3) Purification and Characterization of a β₂-GPI-Specific Ligand

The lipids extracted from each LDL were spotted on a TLC plate anddeveloped in solvent A (FIGS. 3A and 3B). In the plates treated with I₂vapor and molybdenum blue, decreased PC (phosphatidylcholine) andincreased polar forms co-migrating with lysoPC were observed in lipidsderived from oxLDL as compared with derived from of native LDL. Bystaining with orcin/sulfuric acid, pseudo-positive bands were observedat similar Rf positions of Chol, PC, and CL, and others. InLieberman-Burchard reaction, a Chol band was observed for both extractsat almost top, and few bands near the Rf position of CL were observedfor lipid derived from oxLDL. To identify β₂-GPI-specific ligands, thedeveloped plates were subjected to ligand blot in which the plates weretreated with β₂-GPI and anti-β₂-GPI antibodies (FIG. 3B). With all threetested anti-β₂-GPI antibodies (Cof-22, WB-CAL-1, and EY2C9), twopredominant bands and several bands of diffused lipids were stained andobserved at similar Rf positions of CL and glycolipids such asgalactosylceramide (Gal-Cer) and glucosylceramide (Glc-Cer). The bandsdetected in the ligand blot were not stained with a spray of molybdenumblue (FIGS. 3A and 3B).

β₂-GPI-ligand-enriched lipids were scraped from the first TLC plate(hereinafter referred to as “scraped lipids”) (FIG. 3B) and weresubjected to another TLC (developing in solvent B), and the two majorbands were scraped off and subjected to the ligand blot (FIGS. 3C and3D). Two major bands (indicated by arrows in FIG. 3) reacting withβ₂-GPI were named Band-1 (the upper band) and Band-2 (the lower band).

Band-1 was rarely stained in the sequential treatment with β₂-GPI andEY2C9 (or WB-CAL-1) (2-step), but was strongly stained in the case ofthe simultaneous treatment (1-step). In contrast, Band-2 was stainedwell in either treatment with β₂-GPI and EY2C9 (Densitometric analysisin individual experiments indicated significant difference).

The alkaline hydrolyzed (20% NaOH, 100° C., 30 minutes) product ofBand-1 did not bind to β₂-GPI in the ligand blot (data not shown).Band-1 was subjected to reversed phase HPLC by use of solvent C. A majorpeak appeared at 22 min that positively stained in the ligand blot withβ₂-GPI and EY2C9 (FIG. 4), and was attributable to very weakLieberman-Burchard reaction. This peak was designated as oxLig-1. From100 mg protein equivalent of oxLDL, approximately 2.5 mg of oxLig-1 wasrecovered.

(4) Purification of oxLig-2

The oxLig-2, which is a specific ligand to β₂-GPI, was purified from aligand-containing fraction through reverse phase HPLC by use of aSephasil Peptide C18 column (4.6 mm×250 mm, product ofAmersham-Pharmacia Biotech). The thus-separated Band-2 was eluted at aflow rate of 0.5 mL/min by 0.2% acetic acid-containing water (solvent E)and acetonitrile/isopropanol (30/70, v/v) (solvent D) with lineargradation (50–100%) in concentration for 15 minutes and then by 100%solvent D for 15 minutes. The absorbance (A) at 210 nm or at 234 nm wasmonitored. The eluate was fractionated (1 mL/tube) every two minutes.Each fraction was applied to a TLC plate and subjected to ligand blotanalysis using β₂-GPI and EY2C9.

(5) Purification and Properties of oxLig-2

Through HPLC of Band-2, a novel ligand (oxLig-2) was obtained (FIGS. 16Aand 16B).

The peaks corresponding to specific binding to β₂-GPI and EY2C9 wereobserved at about 26.7 min (equivalent to elution volume of 13.4 mL). Inorder to confirm the purification degree of oxLig-2 (fraction 14), thefraction was subjected to HPLC again under the same conditions (FIGS.16C and 16D), and to an LC/MS analysis.

In a positive ionization mass spectrum of oxLig-2, three signals (m/z:383, 441, and 627) were detected (FIG. 17C). Two small peaks; i.e., m/zof 383 (attributable to 7-ketocholesterol) and of 441 (attributable to7-ketocholesterol (+acetone)) are similar to those observed in the caseof ox-Lig-1 and 13-COOH-7-KC (FIGS. 17A, 17C, and 17E).

The signals of 571, 627, and 627 (m/z) shown in the positive ionizationmass spectra of oxLig-1, oxLig-2, and 13-COOH-7KC, respectively, wereidentified as respective parent ions [M+H]+ (FIGS. 17A, 17C, and 17E).The signals of 569, 625, and 625 (m/z) shown in the negative ionizationmass spectra of oxLig-1, oxLig-2, and 13-COOH-7KC, respectively, wereidentified as respective parent ions [M+H]− (FIGS. 17B, 17D, and 17F).In the spectrum of oxLig-2, a signal of 627 (m/z) was identified as aparent ion of dihydro-oxLig-2 (FIG. 17D). In the negative mode, thesignals of 187, 243, and 243 (m/z) shown in the spectra of oxLig-1,oxLig-2, and 13-COOH-7KC, respectively, were identified as respectivefragment ions [D−H]− (FIGS. 17B, 17D, and 17F, and FIG. 18).

In all TLC ligand blot analyses using solvent A or B, the Rf positionwith respect to oxLig-2 is lower than that of the relevant monocarbonylderivatives (oxLig-1 and 13-COOH-7KC). The feature coincides with thepresumed difference in polarity (FIGS. 3, 8, and 15).

In the TLC ligand blot analysis using solvent B, the Rf positions in thebands with respect to oxLig-2 and 13-COOH-7KC methylated by diazomethane(Me-oxLig-2 and Me-13-COOH-7KC, respectively) were higher than that ofthe relevant unmethylated ligands (FIGS. 8 and 15). The peakcorresponding to oxLig-2 (26.7 min) in reverse phase HPLC was observedearlier than oxLig-1 (27.3 min) and 13-COOH-7KC (28.9 min). The peakscorresponding to methylated oxLig-2 and methylated 13-COOH-7KC wereobserved (27.1 min and 30.0 min, respectively) later than those of theunmethylated species. Surprisingly, in TLC ligand blot analyses, theinteraction of β₂-GPI and the anti-β₂-GPI antibodies (Cof-22 and EY2C9)with each ligand was completely suppressed through ligand methylation.

In ELISA of the anti-β₂-GPI antibodies by use of a ligand-coated plate,significant binding of the anti-β₂-GPI autoantibodies (WB-CAL-1 andEY2C9) to solid-phase oxLig-1, oxLig-2, and 13-COOH-7KC were observed,but no such binding was observed with respect to solid-phase methylatedoxLig-2, methylated 13-COOH-7KC, or control lipid (cholesteryllinoleate). Similar results were obtained in the case where a mousemonoclonal anti-β₂-GPI antibody obtained from mice immunized with humanβ₂-GPI was used (Table 1). These three antibodies did not bind to theimmobilized cholesterol or to 7-ketocholesterol. From these results andpreviously reported results [52], it is concluded that the structure ofoxLig-2 is highly likely to be an oxide of cholesteryl linoleate,4,12-dioxo-12-(7-ketocholest-5-en-3β-yloxy)dodecanoic acid (FIG. 18).

TABLE 1 Solid-phase lipid β2-GPI binding (Cof-22 binding) WB-CAL-1binding EY2C9 binding (Ligand) No treatment Methylation No treatmentMethylation No treatment Methylation Cholesteryl 0.061 +/− 0.005 N.T.0.056 +/− 0.002 N.T. 0.074 +/− 0.012 N.T. linoleate oxLig-1 1.307 +/−0.105 N.T. 0.945 +/− 0.068 N.T. 1.458 +/− 0.062 N.T. oxLig-2 1.002 +/−0.084 0.204 +/− 0.029 0.518 +/− 0.023 0.106 +/− 0.018 1.018 +/− 0.1210.076 +/− 0.018 13-COOH-7KC 1.303 +/− 0.049 0.094 +/− 0.002 0.336 +/−0.027 0.077 +/− 0.002 1.062 +/− 0.040 0.052 +/− 0.000 (Means ± SD (n =3) of the absorbance at 490 nm, N.T.: not measured)

FIG. 15 shows the results of ligand blot with respect to oxLig-2,Me-oxLig-2, 13-COOH-7KC, and Me-13-COOH-7KC. As is clear from FIG. 15,reactivity of β₂-GPI and that of the β₂-GPI antibody is lost throughmethylation of oxLig-2 and 13-COOH-7KC.

oxLig-1 was further analyzed through LC/MS. The intensities of bothpositive and negative ions attributed to oxLig-1 were detected for themain peak (at 8.3 min) at 234 nm (FIG. 5A). A positive ionization massspectrum gave a signal at m/z 571, which was considered to be attributedto (M+H)⁺, and at m/z 383 (FIG. 5D), which was identical to thatattributed to ionized 7-ketocholesterol (FIG. 5B).

In the negative ion mode, a signal of fragment ion was detected at m/z187 (FIG. 5C). oxLig-1 was treated with diazomethane in diethyl ether togive methylated oxLig-1, which was analyzed through LC/MS. MethylatedoxLig-1 was eluted later than oxLig-1 (FIG. 5A, lower), and signals ofthe largest fragment ion (9.0 min) was detected at m/z 585 and at m/z201, in the positive and negative ion modes, respectively (FIGS. 5E and5F). These data are consistent with the structure of9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid.

(6) Synthesis and Analysis of oxLig-1

In order to confirm the structure of oxLig-1, oxLig-1 was synthesizedfrom 7-ketocholesterol and azelaic acid. As shown in FIG. 6, thematerials were processed with WSC and DMAP in acetone at roomtemperature for two days, whereby both materials were conjugated eachother. The product was isolated through column chromatography on silicagel. The structure of the synthesized oxLig-1 was verified to purifiedoxLig-1 through ¹H- and ¹³C-NMR spectroscopy and FD mass spectrometry.In the ¹H-NMR spectrum of synthesized oxLig-1 (synthesized oxLig-1)(FIG. 7A), the signals of H-3 and H-6 are shown at δ 4.69–4.78 and 5.71ppm as multiplet and singlet, respectively. Furthermore, three peaksassignable to carbonyl carbons are shown at δ 202.5, 179.7, and 173.4ppm together with two signals of olefin carbons at δ 164.5 and 127.1 ppm(FIG. 7B).

The synthesized oxLig-1 was then esterified with diazomethane in diethylether to give methylated oxLig-1. In FIGS. 7C and 7D, by ¹H- and ¹³C-NMRspectra of synthesized oxLig-1 are shown. The new singlet was observedin its ¹H-NMR spectrum at δ 3.67 ppm, strongly suggesting that oxLig-1has a carboxyl group (FIG. 7C). Then, synthesized oxLig-1 was subjectedto TLC and ligand blot analysis with β₂-GPI and anti-β₂-GPI antibodies.Synthesized oxLig-1 showed the same Rf position and bindingcharacteristics to Cof-22, WB-CAL-1, and EY2C9 antibodies, as previouslydescribed for oxLig-1 derived from oxLDL (FIGS. 3 and 8).

The synthesized oxLig-1 and its methylated compound were furtheranalyzed through LC/MS. The LC chromatograms and mass spectra of bothcompounds (FIG. 9) were identical to the corresponding compounds derivedfrom oxLDL (FIG. 5). In FIG. 9D, the small peak at 8.3 min is attributedto synthesized oxLig-1 that remained underivatized after the methylationreaction. The underivatized material was identified as synthesizedoxLig-1 through mass spectrometry.

(7) Liposome Binding to Macrophages

Binding to the J774A.1 cells of exogenous PS-Chol-liposomes increaseddepending on the amount of DPPS (FIG. 10). In contrast, binding to thecells of oxLig-1-Chol-liposomes was relatively low. Similar bindingprofiles were obtained with Chol-free liposomes of PS or oxLig-1. Whenmouse peritoneal macrophages were used in place of J774A.1 cells,comparable liposome binding was observed.

(8) Antibody-Dependent Liposome Binding to Macrophages

Binding (4° C., 2 hours) of PS-liposomes to oxLig-1-liposomes increaseddramatically upon simultaneous addition of β₂-GPI and WB-CAL-1, and wasalso dependent on the concentration of WB-CAL-1 (FIGS. 11A and 11B). Inthe same assay, subclass-matched control antibodies had no effect onsuch binding. The uptake (37° C., 5 hours) of oxLig-1-liposomes byJ774A.1 cells increased significantly by incubating (4.36 pmol[³H]DPPC/mg protein) with β₂-GPI and WB-CAL-1, as compared to incubationwithout β₂-GPI and WB-CAL-1 (0.72 pmol [³H]DPPC/mg protein) (data notshown). As shown in FIG. 11C, binding of synthesized oxLig-1-liposomesto the macrophages also increased depending on the ligand concentrationin liposomes. In the case of synthesized oxLig-1, the binding reachedalmost plateau at the concentration of 10 mL %.

(9) Binding of Liposomes Containing oxLig-2, 13-COOH-7KC, or the Like toMacrophage

Direct binding of liposomes containing oxLig-1, oxLig-2, or 13-COOH-7KCto macrophages, J774A.1 cells, was compared with that of liposomescontaining DPPS. DPPS-containing liposomes exhibited binding to themacrophages with ligand concentration dependency. In contrast, bindingof liposomes containing oxLig-1, oxLig-2, or 13-COOH-7KC to macrophageswas relatively low or negligible small (FIG. 19). As is clear from FIG.19, scavenger receptors are not related to binding of these liposomes tomacrophages except the case of DPPS-containing liposomes. In otherwords, uptake of liposomes containing oxLig-1, oxLig-2, or 13-COOH-7KCto J774A.1 cells was significantly enhanced in the presence of bothβ₂-GPI and an anti-β₂-GPI antibody (WB-CAL-1), as compared withcholesteryl linoleate-liposomes (control) (FIGS. 20A, 20B, 20C, and20D). In contrast, substantially no binding of liposomes was observedafter methylation of oxLig-2 or 13-COOH-7KC (FIGS. 20C and 20D).

(10) Detection of Autoantibodies in APS Patients with Episodes ofArterial Thrombosis through ELISA Using a β₂-GPI-Synthesized oxLig-1Complex.

As shown in FIG. 12, autoantibodies against a complex of β₂-GPI andsynthesized oxLig-1 were detected in APS patients at high frequency.There was a good correlation between values of autoantibodies againstthe complex antigen and those of β₂-GPI-dependent aCL or anti-β₂-GPIantibodies. So far as the tests were preformed, such autoantibodiesderived from APS cross-reacted with β₂-GPI complexed with CL or oxLig-1,but not that complexed with oxidized PAPC (oxPAPC) (Table 2, Exp-1). Theantibody binding did not correlate with the amount of TBARS in lipid.Further, the antibody binding was provided only by the interactionbetween oxLig-1 and intact β₂-GPI (Exp-2). In contrast, no antibodybinding was provided by nicked β₂-GPI or haptoglobin having “sushidomains” (which did not have PL binding property).

TABLE 2 Experiment 1 Solid-phase lipid Binding of β₂-GPI-dependent aCL(Absorbance, mean ± SD, n = 3) (TBARS)^(a) Cof-22 EY2C9 WB-CAL-1APS-1^(b) APS-2^(b) CL (4.46) 1.132 ± 0.025 1.269 ± 0.014 1.361 ± 0.0082.099 ± 0.216 1.282 ± 0.041 Synthesized 1.066 ± 0.114 0.915 ± 0.0721.035 ± 0.062 1.130 ± 0.177 1.222 ± 0.057 oxLig-1 (0.66) PAPC (107.8)0.019 ± 0.003 0.011 ± 0.003 0.012 ± 0.001 0.000 ± 0.001 0.010 ± 0.002oxPAPC^(c) (218.3) 0.073 ± 0.007 0.009 ± 0.002 0.015 ± 0.005 0.007 ±0.002 0.015 ± 0.001 Experiment 2 Binding of protein-dependent oxLig-1antibody (Absorbance, mean ± SD, n = 3) Added protein Cof-22 EY2C9WB-CAL-1 APS-1^(b) APS-2^(b) w/o 0.050 ± 0.002 0.005 ± 0.001 0.005 ±0.002 0.012 ± 0.003 0.014 ± 0.001 β₂-GPI 1.007 ± 0.031 1.147 ± 0.0280.812 ± 0.023 1.043 ± 0.054 0.755 ± 0.024 Nicked β₂-GPI 0.164 ± 0.0070.005 ± 0.001 0.003 ± 0.001 0.008 ± 0.003 0.011 ± 0.001 Haptoglobin0.049 ± 0.001 0.006 ± 0.001 0.006 ± 0.001 0.018 ± 0.011 0.015 ± 0.002Ovalbumin 0.042 ± 0.001 0.004 ± 0.002 0.006 ± 0.002 0.009 ± 0.004 0.016± 0.001

Table 2 shows the results of binding of anti-β₂-GPI antiphospholipidantibodies (Abs) to lipid-proptein complexes observed throughenzyme-linked immunosorbent assay (ELISA).

Data shown in Experiment 1 represent binding of antibodies in thepresence of β₂-GPI (15 μg/mL), and data shown in Experiment 2 representthe results of ELISA performed by use of a synthesizedoxLig-1-solidified plate in the presence of each of the proteins (15μg/mL) shown in Table 2. The amount of TBARS (thiobarbituric acidreactive substance) shown in the Table is based on nmol (malondialdehydeequivalent)/mg (lipid). APS-1 and APS-2 were obtained from serum samplesof APS patients having an episode of arterial thrombosis with removal ofβ₂-GPI. oxPAPC was obtained by exposing PAPC in air at room temperaturefor 24 hours. Nicked β₂-GPI was prepared through plasmin treatment.[43].

(11) The Following Results were Obtained through Further Analysis of theRelationship Between the anti-β₂-GPI-oxLig-1 Antibody Value and ClinicalObservations.

When the antibody value (OD) of a plasma sample exceeded the averaged(30 healthy volunteers) antibody value by more than 3×SD (standarddeviation), the sample was regarded to be positive to a specificantibody (as a cut-off value).

The IgG anti-β₂-GPI-oxLig-1 antibody was observed in 73% theinitial-stage APS patients (35/48); in 59% the APS patients with SLE(second-stage APS) (23/39); and in 11% the solo-SLE patients (5/46)(Table 3).

TABLE 3 Patient No. % SLE only 46 APS 87 Primary 48 55 Secondary 39 45Clinical profile Thrombosis 75 56 Arterial thrombosis only 27 20 Venousthrombosis only 29 22 Arterial and venous thrombosis 19 14 Pregnancymorbidity 32/120 27 Thrombocytopenia 24/128 19 Autoantibodyβ₂-GPI-dependent aCL 77/133 58 (anti-β₂-GPI-CL antibody) Anti-β₂-GPIantibody 48/133 36 Anti-β₂-GPI-oxLig-1 antibody 63/133 47 Lupusanticoagulants 62/113 55

In all patients (133 patients) investigated in the present example, theanti-β₂-GPI-oxLig-1 antibody value was found to be strongly correlatedwith the antibody value of the β₂-GPI-dependent aCL (anti-β₂-GPI-CLantibody) and that of the anti-β₂-GPI antibody (correlation factor r²:0.72 and 0.81, respectively) (FIG. 14).

In these patients (133 APS and/or SLE patients), significantrelationship was observed between the anti-β₂-GPI-oxLig-1 antibody valueand episodes of thrombosis (arterial and/or venous thrombosis, arterialthrombosis, and venous thrombosis) or pregnancy morbidity, but nosignificant relationship was observed between the antibody value andthrombocytopenia (p=4.2×10⁻⁸ (Fisher's exact test); Odds ratio 8.15,p=1.7×10⁻⁷; 8.00, p=0.018; 2.29, P=0.0077; 3.03, and P=0.31; 1.38,respectively) (Table 4). Assay sensitivity, specificity, and expectedvalues with respect to arterial thrombosis diagnosis were remarkablyexcellent as compared with diagnosis of venous thrombosis, pregnancymorbidity, and thrombocytopenia (Table 4).

TABLE 4 Thrombosis Arterial thrombosis Venous thrombosis Pregnancymorbidity Thrombocytopenia Auto- Odds Odds Odds Odds Odds antibody + −p* ratio + − p* ratio + − p* ratio + − p* ratio + − p* ratio Positive 5112 4.2 × 10⁻⁸ 8.15 36 27 1.7 × 10⁻⁷ 8.00 29 34 0.018 2.29 21 34 0.00773.03 13 48 0.31 1.38 Negative 24 46 10 60 19 51 11 54 11 56 (NS)Specificity 0.79 0.69 0.60 0.61 0.54 Sensitivity 0.68 0.78 0.60 0.630.54 Expected 0.73 0.72 0.60 0.63 0.54 Value +: Presence −: Absence p*:Fisher exact test, NS: No significant difference

In addition, as compared with the antibody value of the β₂-GPI-dependentaCL and that of the anti-β₂-GPI antibody, the anti-β₂-GPI-oxLig-1antibody value was more strongly correlated with thrombosis (arterialand/or venous thrombosis) (p=1.5×10⁻⁶ (Fisher's exact test); Odds ratio6.02, p=5.2×10⁻⁵; 4.93, respectively).

The anti-β₂-GPI-oxLig-1 antibody values of the patients having anepisode of thrombosis (arterial and/or venous thrombosis, arterialthrombosis, or venous thrombosis) or a pregnancy-related disease wassignificantly higher than that of subjects having no such an episode(Mann-Whitney U-test, p<0.0001, p<0.0001, P=0.025, and P=0.011) (FIG.15). In contrast, no relationship was observed between the presence ofthe antibody or the antibody value and the episode of thrombocytopenia(Mann-Whitney U-test, p=0.45).

<3> Discussion

A strong evidence for specific binding interactions among β₂-GPI, oxLDL,and an anti-β₂-GPI autoantibody, was obtained by use of an opticalbiosensor. A ligand specific to β₂-GPI was purified, and its structureand involvement in macrophage uptake of oxLDL was characterized by usinga synthesized ligand. The structure of the ligand was confirmed byreproducing its properties with chemically synthesized9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid.

It appears that oxidation of LDL plays an important role inatherogenesis [44]. To examine mechanisms related to the development ofatherosclerosis, several experimental models of denatured LDL, such asMDA-LDL, acetylated LDL, and CuSO₄-mediated oxLDL (CuSO₄-oxLDL), havebeen used. Among these LDLs, Cu²⁺-ion can induce LDL oxidation,resulting in highly reproducible LDL damage [45]. This process leads toproduction of oxLDL that shares many structural and functionalproperties with LDL oxidized by cells or LDL extracted from arterialatherosclerotic plaques. Incubation of LDL with several different typesof cells or incubation of LDL with Cu²⁺-ion even in the absence of cellsresult in generating oxidatively modified LDL with similar properties[46]. There is general agreement about the use of CuSO₄-oxLDL as anautoantigen because oxLDL has been found in atheromatous lesions andoxLDL extracted from atherosclerotic lesions exhibits nearly all of thephysicochemical and immunological properties of CuSO₄-oxLDL [24].

Antibodies against oxLDL recognize substances in atherosclerotic lesionsthat are not present in normal arteries. It has been reported that aCLraised in SLE patients cross-reacted with MDA-LDL [27], while otherresearch groups found that another population of anti-oxLDL antibodiesreacted to oxidized PC (such as POVPC)-protein adducts in LDL molecules[33, 47].

However, recent studies elucidated that TBARS generation was notconsistent with β₂-GPI binding to CuSO₄-oxLDL [48].

As MDA is a hydrophilic short-chain aldehyde, it readily diffuses awayfrom LDL particles [49]. It was also observed that, after dialysis,TBARS in the oxLDL preparations decreased to undetectably low levels. Inthis study, CuSO₄-oxLDL showed highly specific binding to immobilizedβ₂-GPI (FIG. 1A). The weak binding of native LDL to β₂-GPI might reflectthe binding of β₂-GPI to lipoprotein [a], which is composed of LDL andapolipoprotein [a] (apo[a]), as described in [50]. However, suchinteraction between native LDL and β₂-GPI may not expose suitableepitopes to anti-β₂-GPI autoantibody (FIG. 1B). Further, highly specificbinding of anti-β₂-GPI autoantibody, two monoclonal antibodies, andantibodies in two typical anti-β₂-GPI positive serum samples (derivedfrom APS patients with episodes of arterial thrombosis) was onlyobserved in the presence of the complex of β₂-GPI and the lipid ligandderived from CuSO₄-oxLDL (FIGS. 2, 3, and 12, Table 2). Oxidativemodification of LDL actually includes a series of complex changes suchas lipid peroxidation, modification of side chains of amino acids byactive aldehydes, increased surface charge, and polymerization.

Among diverse modified molecules in oxLDL, β₂-GPI obviously bound to acomponent in the lipid moiety.

Linoleic acid is a predominant polyunsaturated fatty acid in LDL and ispresent mainly as Chol-ester [51].

In mildly oxidized LDL, cholesteryl hydroperoxyoctadecadienoate(Chol-HPODE) and cholesteryl hydroxyoctadecadienoate (Chol-HODE) weredetected as main constituents of oxidation products [52]. Chol-HPODE hasbeen reported to inactivate platelet-derived growth factor [53].7-Hydroxycholesterol (both free and esterified) is the major oxysterolformed in an earlier stage of LDL oxidation, with 7-ketocholesteroldominating at later stages [54]. Recent studies indicated that elevatedplasma levels of 7β-hydroxycholesterol may be associated with anincreased risk of atherosclerosis [55]. At later stages in LDLoxidation, cholesteryl esters or 7-ketocholesteryl esters of9-oxononanoate derived from cholesteryl linoleate [56] were detected asthe most abundant fraction of oxidized cholesteryl linoleate [57, 58].Cholesteryl ester core-aldehydes react with the free ε-amino group oflysines, form complexes with proteins, and were evident in humanatheroscleorotic lesions [58, 59]. In the present study, oxLig-1 wasidentified to be 9-oxo-9-(7-ketochlest-5-en-3β-yloxy)nonanoic acid, oneof the oxidation products of cholesteryl linoleate, and a major ligandfor β₂-GPI. However, it still remains to be elucidated whether9-oxo-9-(7-ketochlest-5-en-3β-yloxy)nonanoic acid is actually present inbiological samples as an oxLDL ligand.

Chemically modified LDL can be rapidly taken up by macrophages viareceptor-mediated endocytosis, resulting in foam cell formation [44]. Asmodels of oxLDL, oxLig-1- and PS-liposomes were used to study theirbinding to J774A.1 cells (FIG. 7). PS-liposomes bound to macrophages viascavenger receptor(s), while oxLig-1 did not seem to be a major ligandfor scavenger receptors. However, the binding of oxLig-1-liposomes toJ774A.1 cells at 4° C. increased up to 14 times when oxLig-1-liposomeswere added simultaneously with β₂-GPI and WB-CAL-1. The uptake ofoxLig-1-liposomes with J774A.1 cells at 37° C. also significantlyincreased by incubation with β₂-GPI and WB-CAL-1. This binding anduptake might be mediated by the Fcy receptor. The uptake by macrophagesof immune complexes containing oxLDL through the Fcγ type I receptortransformed macrophages into foam cells [60, 61], and could acceleratethe atherogenic process [62–64].

Autoantibodies against a solid phase oxLig-1 complexed with β₂-GPI weredetected in serum samples collected from APS patients having episodes ofarterial thrombosis (FIG. 12). Further, there was a good correlationbetween anti-β₂-GPI-oxLig-1 antibody value, anti-β₂-GPI antibody value,and β₂-GPI-dependent aCL titer. In contrast, it has been reported thatsome aPL recognize adducts of oxidized PLs and β₂-GPI [31]. It has notbeen determined whether oxLig-1can form covalent adducts with β₂-GPI,and the possibility can not yet be excluded. However, it is clear thatinteraction between oxLig-1 and β₂-GPI is essential to expressantigenicity for the autoantibodies shown in Table-2, Exp-2. It was alsosuggested that domain V, which contains the- PL-binding region [15, 16],is distinguished from domains in which epitopes, recognized by aPL inAPS patients, locate [38, 65].

In addition to promoting lipid deposition in macrophages, oxLDL isconsidered to have another characteristic that it may accelerateatherogenesis. oxLDL is chemotactic for monocytes and for T cells [66,67] and is cytotoxic for cultured endothelial cells [68]. It has beenalso reported that peroxisome proliferator-activated receptor γ (PPARγ),a transcriptional regulator of genes linked to lipid metabolisms, isactivated by components of oxLDL, such as 9-HODE, and 13-HODE and CD36,a scavenger receptor, is up-regulated by a combination of PPARγ andretinoid X receptor ligands [69]. PPARγ enhances the uptake of oxLDL,thereby promoting foam cell formation. It remains to be determined ifoxLig-1 has any specific influence on nuclear receptors or otherbiological functions such as intracellular signal transductions.

In summary, a ligand for β₂-GPI present in oxLDL was isolated andcharacteirzed. The ligand (oxLig-1), i.e.,9-oxo-9-(7-ketochlest-5-en-3β-yloxy)nonanoic acid, mediates liposomeuptake by macrophages in the presence of β₂-GPI and an anti-β₂-GPIautoantibody. These findings on the ligand provide a specific structuraland mechanistic link between β₂-GPI and anti-β₂-GPI autoantibodies andatherogenesis in APS.

The ω-carboxyl group introduced through oxidation by Cu²⁺ was indicatedto play an important role in interaction between β₂-GPI and the ligand.The ligand, oxLig-2, was identified as4,12-dioxo-12-(7-ketocholest-5-en-3β-yloxy)dodecanoic acid (FIG. 18).

Although results of methylation of oxLig-2 indicated that the carboxylicgroup was present in the acyl chain, the accurate position of theketonic moiety cannot be determined through mass spectroscopy. However,since cholesteryl linoleate is one of the important cholesteryl estersof LDL [52], the positions of the ketonic moieties are highly likely9-position and/or 13-position.

β₂-GPI did not bind to cholesterol, 7-ketocholesterol, or cholesteryllinoleate, but did form significant bonding to oxLig-1, oxLig-2, or13-COOH-7KC. Thus, the formed oxysterol esters having a carboxylatedlong acyl chain constitute a novel class of amphipathic ligands suitablefor β₂-GPI. Furthermore, the fact that methylation of these ligandsinhibits interaction between the ligands and β₂-GPI indicatedrequirement of a free carboxylic group for structure recognition.

The results of experiments indicated that oxidized cholesteryl esters,particularly such esters having 7-ketocholesterol and a carboxyl groupin the acryl group functioned as ligands to β₂-GPI and an anti-β₂-GPIautoantibody. One predominant biologically oxide compound originatingfrom plasma LDL may be a ω-carboxylated oxysterol such as oxLig-1 oroxLig-2. 13-COOH-7KC, which is an artificially synthesized compound,also formed significant bonding to β₂-GPI, similar to the cases ofoxLig-1 and oxLig-2.

<4> Fabrication of the Kits of the Present Invention

The kit 1 of the present invention containing the following elements wasprepared:

-   1. A 96-well immuno-plate on which oxLig-1 has been immobilized (1    sheet);-   2. β₂-GPI standard solution (1 set);-   3. Anti-β₂-GPI antibody (WB-CAL-1) (1 vial);-   4. HRP-labeled anti-mouse IgG antibody (1 vial);-   5. o-Phenylenediamine solution (1 vial);-   6. Aqueous hydrogen peroxide (1 vial); and-   7. Reaction-terminating liquid (1N HCl) (1 vial).

The kit 2 of the present invention containing the following elements wasprepared:

-   1. A 96-well immuno-plate on which oxLig-1 has been immobilized (1    sheet);-   2. β₂-GPI (1 vial);-   3. HRP-labeled anti-human IgG antibody (1 vial);-   4. Tetramethylbenzidine solution (1 vial);-   5. Aqueous hydrogen peroxide (1 vial); and-   6. Reaction-terminating liquid (1N HCl) (1 vial).    <5> References-   15. J. immunol. 152: 653–659-   16. EMBO J. 18: 5166–5174-   24. J. Clin. Invest. 84: 1086–1095-   27. Lancet. 341: 923–925.-   31. J. Clin. Invest. 98: 815–825-   33. J. Clin. Invest. 103: 117–128-   35. J. Immunol. 148: 3885–3891-   36. Arthritis Rheum. 37: 1453–1461-   37. J. immunol. 149: 1063–1068-   38. Blood. 87: 3262–3270-   39. J. Clin. Invest. 43: 1345–1353-   40. Anal. Biochem. 95: 351–358-   41. J. Biol. Chem. 226: 497–509-   42. J. Biol. Chem. 265: 5226–5231-   43. Int. Immunol. 12: 1183–1192-   44. J. Biol. Chem. 272: 20963–20966-   45. Clin. Chem. 38: 2066–2072-   46. Eur. Heart J. 11 (Suppl. E): 83–87-   47. J. Biol. Chem. 269: 15274–15279-   48. Lupus. 7 (Suppl. 2): 135–139-   49. J. Lipid Res. 28: 495–509-   50. Blood. 90: 1482–1489-   51. Arterioscler. Thromb. Vasc. Biol. 15: 1131–1138-   52. Anal. Biochem. 213: 79–89-   53. J. Biol. Chem. 273: 19405–19410-   54. J. Lipid Res. 38: 1730–1745-   55. Atherosclerosis. 142: 1–28-   56. FEBS Lett. 304: 269–272-   57. J. Lipid Res. 36: 1876–1886-   58. J. Lipid Res. 38: 1347–1360-   59. J. Lipid Res. 39: 1508–1519-   60. J. Exp. Med. 168: 1041–1059-   61. Atherosclerosis. 135: 161–170-   62. Arterioscler. Thromb. 12: 1258–1266-   63. Arterioscler. Thromb. Vasc. Biol. 15: 990–999-   64. J. Lipid Res. 36: 714–724-   65. Proc. Natl. Acad. Sci. USA 95: 15542–15546-   66. Proc. Natl. Acad. Sci. USA 84: 2995–2998-   67. J. Clin. Invest. 92: 1004–1008-   68. Atherosclerosis 3: 215–222-   69. Cell. 93: 229–240

INDUSTRIAL APPLICABILITY

The derivative of the present invention specifically binds to β₂-GPI,and thus can be used for, for example, detecting or purifying β₂-GPI.The derivative can also be applicable to the solid phase of the presentinvention, the assay method of the present invention, the detectionmethod of the present invention, and the assay kit of the presentinvention. Thus, the derivative of the present invention is remarkablyuseful. Furthermore, oxLDL can be assayed making use of the derivativeof the present invention through a competitive method and the derivativecan be employed as a standard substance for oxLDL assay.

The solid phase of the present invention can be used, for example, insimple, quick detection of β₂-GPI or purification of β₂-GPI as well asis applicable to the assay method of the present invention, thedetection method of the present invention, and the assay kit of thepresent invention. Thus, the solid phase of the invention is remarkablyuseful.

Through the assay method 1 or 2 of the present invention, β₂-GPI, anautoantibody against a complex of β₂-GPI and the derivative of thepresent invention, or a similar substance can be assayed quickly in asimple manner. Thus, the assay methods 1 and 2 of the present inventionare remarkably useful.

Through the detection method of the present invention, a disease can bedetected quickly in a simple manner. Thus, the detection method of thepresent invention is remarkably useful.

By use of the assay kit 1 or 2 of the present invention, the assaymethod 1 or 2 can be carried out more quickly and in a simpler manner.Thus, the assay kits of the present invention are remarkably useful.

The invention claimed is:
 1. A cholesterol derivative represented by thefollowing formula (3).


2. A cholesterol derivative represented by the following formula (4).


3. A cholesterol derivative represented by the following formula (7).


4. An assay method for β₂-GPI, characterized in that the method includesat least the following steps: a step of forming a complex of β₂-GPI anda cholesterol derivative immobilized on a solid phase by bringing aspecimen into contact with the solid phase (Step 1), wherein thecholesterol derivative is represented by the following formula (3), (4)or (7):

and a step of detecting β₂-GPI contained in the complex which has beenformed in Step 1 (Step 2).